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8.3 Medical significance of Rauwolfia 115
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8.2.3 Rauwolfia serpentina (L.) Benth. ex. Kurz.
R. serpentina (L.) Benth. ex. Kurz. is an under shrub, belongs to Apocynaceae family of dicotyledonous. It is indigenous to India and other tropical countries of Asia
and is naturalized in distribution. Typical morphological characteristics of the plant
include small in appearance, completely erect in size with presence of glabrous
shrub, whe re the approximate height includes 30e60 cm. Also the presence of
whorled type leaves possess length 7.5e17.5 cm, with lanceolate or oblanceolate
appearance in shape, acute or acuminate aperture, along with characteristic tapering
at the petiole. Moreover, the plant bears flowers with white to pinkish appearance,
peduncles of 5.0e7.5 cm in length, pedicels and calyx red, with calyx lobes of
2.5 mm long and lanceolate. The roots of the plants are 5e15 cm long and
3e20 mm in diameter, subcylindrical to tapered structure. Moreover, the plants
include tortuous or curved shaped structure, rarely branched appearance, occasionally bearing twisted rootlets. Moreover, the external appearance of the plant indicates light brown to grayish yellow and grayish brown color.
8.3 Medical significance of Rauwolfia
Plants are utilized therapeutically in different countries and are a source of numerous
potent and powerful drugs. Medicinal plants are used by 80% of the world’s population as the main accessible medicines especially in developing nations. Rauwolfia
can be regarded as a typical drug of ayurvedic medicaments.
R. serpentina has a broad range of therapeutic spectrum, mainly effective in the
treatment of hypertension and psychotic disorders like schizophr enia, anxiety, epilepsy, insomnia, insanity, and furthermore, utilized as a sedative, a hypnotic drug.
Rauwolfia has been studied widely in researches as a treatment for autistic children
between the ages of 3.5 and 9 years (Lehman et al., 1957).
The plant is accounted for a large number of therapeutically useful indole alkaloids and these alkaloids are extensively situated in the roots. Alkaloids of this plant
have a great therapeutic significance to treat cardiovascular diseases, hypertension
(Silja et al., 2008), arrhythmia, breast cancer, and human promyelocytic leukemia
(Itoh et al., 2005).
The Rauwolfia root has been consumed since the pre-Vedic period as a medication in India, to treat snake bites and fever and bug stings (Thakar, 2010). Its roots
are utilized as an esteemed medicine for blood pressure, insomnia anxiety, excitement, schizophrenia, insanity, epilepsy, hypochondria, and other disorders of the
central nervous system (Singh et al., 2010; Agrawal and Mishra, 2013).
The root was believed to stimulate uterine contraction and suggested for the use
in childbirth. However, the juice of the leaves has been used as a remedy for the
opacity of the cornea. Rauwolfia’s juice and extract acquired from the root can be
used for treating gastrointestinal and circulatory illnesses. The juice of tender leaves
and root extracts are used to treat liver pain, stomach pain, dysentery, and to eliminate intestinal worms.
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The extract is likewise utilized to treat cancer which is one of the leading causes
of death. Plant extract has been reported to use in treatment of prostate cancer and
AIDS (Dey and De, 2011). Extracts from the root and bark of the plant are enriched
with compounds of b-carboline alkaloid family of which the main constituent is
alstonine. This compound has been reported to reduce tumor cell growth in mice
inoculated with YC8 lymphoma cells or Ehrlich ascetic cells. The plant extract
has antiprostate cancer activity in both in vitro and in vivo model systems which,
based upon analyses of gene expression patterns of treated prostate cancer cells,
may be modulated by its effects on DNA damage and cell cycle control signaling
pathways.
8.4 Phytochemical constituents of Rauwolfia
The phytochemical analysis of R. serpentina has numerous medicinal values. Alkaloids are huge cluster of phytochemicals which contain a heterocyclic nitrogen ring.
Till date, over 6000 basic nitrogen containing organic compounds have been isolated,
which are now classified under different categories of alkaloids. Out of this huge number, around 15% of compounds have been isolated from vascular terrestrial plants of
150 different families. The pure alkaloids are used as analgesic, antispasmodic, and
bactericidal effects (Okwu and Okwu, 2004). The medicinal value of plants lies in
the bioactive phytochemical constituents that produce definite physiological effects
on human body. These natural compounds formed the base of modern drugs as we
use today (Koche et al., 2010). Herbal medicines are becoming popular in modern
world as people resort to natural therapies. Natural products isolated from higher
plants and microorganisms have been providing novel clinically active drugs (Nirai-
mathi et al., 2012). The plant contains more than 70 distinct alkaloids which belong
to the monoterpenoid indole alkaloid (MIA) family. The major alkaloids are reserpine,
ajmaline, ajmalicine, ajmalimine, deserpidine, indobine, indobinine, reserpiline,
rescinnamine, rescinnamidine, serpentine, serpentinine, and yohimbine (Howes and
Louis, 1990; Srivastava et al., 2006). Chemical structures of major alkaloids present
in R. serpentina are presented in Fig. 8.1. Different types of alkaloids in
R. serpentina along with their physical and medicinal properties are given in Tab l e 8.2 .
8.4.1 Reserpine
Reserpine is a pure crystalline single alkaloid. It is a white-to-yellow powder that
becomes darker when exposed to light. It is odorless, insoluble in water, slightly soluble in alc ohol, and freely soluble in acetic acid. It has a chemical formula of
C
33H40N2O9
trimethyl benzoic acid ester of reserpic acid, an indole derivative of 18-hydroxy
yohimbine type) is used in hypersensitive reactions and also act as natural tranquillizer (Banerjee and Modi, 2010). Reserpine can be used in the antihypertensive actions by act on peripheral nervous system by binding to catecholamine storage
, a molecular mass of 609 g, and a bitter taste. Reserpine (3,4,5-
8.4 Phytochemical constituents of Rauwolfia 117
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FIGURE 8.1
Chemical structures of some alkaloids present in Rauwolfia serpentina.
vesicles present in the nerve cell (Ellenhorn and Barceloux, 1988; Gilman et al.,
1990). It is useful for the treatment of hypertension, cardiovascular diseases, and
neurological diseases (Weiss and Fintelmann, 2000; Pullaiah, 2002).
The mechanism of action of reserpine is well researched and well documented.
Reserpine binds to protein receptors called vesicular monoamine transporters
(VMATs) in the organelle membranes of specialized secretory vesicles of presynaptic neurons. Reserpine prevents intracellular neurotransmitters from binding to
VMAT proteins and stops secretory vesicles from uptaking neurotransmitters. Ultimately, use of reserpine provides that no or few neurotransmitters are released from
the presynaptic neuron (Nammi et al., 2005). As a result, no or only slight promulgation of the nerve impulse occurs in the postsynaptic neuron.
8.4.2 Biosynthesis of reserpine
Alkaloids have a strong geneticephysiological function and background in the organisms which produce them. The biogenesis of alkaloids is therefore a part of
the total geneticefunctional strategy of such metabolisms. In the genus Rauwolfia,
MIAs are formed via complex biosynthetic sequences. The characteristics of
R. serpentina are only the most prominent representatives of the aforementioned
class of specialized metabolites, collectively forming several hundred different
MIA structures within the plant genera. MIAs are medicinally important class of
compounds abundant in the roots of Rauwolfia species (Apocynaceae). MIAs
have the pentacyclic ring system consisting of monoterpene and indole moieties
which are biologically derived from secologanin and tryptophan, respectively.
MIAs are widespread within the Apocynaceae, underscoring the importance of
numerous members of the botanical family as sources of high-value compounds
boasting pharmacological potential.
Table 8.2 Phytochemicals constituents of Rauwolfia and their medicinal properties.
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Alkaloids
Reserpine C
Ajmaline C
Rescinnamine C
Serpentine C
Deserpidine C
Yohimbine C
Ajmalicine C
Molecular
formula Nature Functions
33H40N2O9
Indole alkaloid,
soluble in
Antipsychotic,
antihypertensive
chloroform
20H26N2O2
Alkaloid, miscible in
Antiarrhythmic 158C Indole alkaloids antiarrhythmics
water
35H42N2O9
Weakly basic indole
Antihypertensive 238C Alkaloid angiotensin converting
alkaloids
20H21N2O3
Basic anhydronium
Tranquilizer 153C Type II topoisomerase inhibitor Dassonneville
alkaloids
32H38N2O8
Ester alkaloid Antipsychotic
and
antihypertensive
21H26N2O3
Indoloquinolizidine
alkaloid
Selective alphaadrenergic
antagonist,
aphrodisiac
21H24N2O3
Indoline alkaloids Vasodilator 250C Vasodilator agent,
Melting
point Category of drug References
264.5C Indole alkaloids
antihypertensive agent,
adrenergic uptake inhibitor,
antipsychotic agent,
hypotensive agent, membrane
transport modulator,
neurotransmitter agent (OCT2
substrates)
(class I and III), cardiovascular
system, membrane transport
modulators, secologanin
tryptamine alkaloids, sodium
channel blocker, voltage-gated
sodium channel blocker
enzyme inhibitor cardiovascular
system
230.5C Inhibitor of the ATP/Mg
pump
241C Treatment of erectile
dysfunction
antihypertensive agent
118 CHAPTER 8 Rauwolfia serpentina
Weiss and
Finelman
(2000),
Pullaiah (2002),
Nammi et al.
(2005),
Banerjee and
Modi (2010)
Brugada et al.
(2003)
Kolh et al.
(1954)
2þ
et al. (1999)
Varchi et al.
(2005)
Morales (2000)
Wink and
Roberts (1998)
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The shikimate pathway consists of a sequence of seven metabolic steps, in which
phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the
precursor of the aromatic amino acids and many aromatic secondary metabolites.
Tryptophan decarboxylase catalyzes the conversion of
Common to the biosynthesis of all MIAs is the formation of their terpenoid precursor, secologanin intermediate of MEP pathway (Stockigt and Zenk, 1977). Its subsequent ligation to tryptamine yields the universal intermediate, Strictosidine. There
are different alkaloids produced due to enzyme strictosidine synthase (STR) in
R. serpentina, mainly the indole alkaloids are shown in Fig. 8.2. The gener al role
played by intermediate strictosidine in the biosynthesis of all MIAs is firmly established (Kutchan et al., 1988; Bracher and Kutchan, 1992).
Shikimatic pathway MEP Pathway
L-tryptophan to L-tryptamine.
Reserpine
1,2-
b-(R)-Dihydrovomilenine
DHVR
Tryptamine +
Strictosidine
Vomilenine
VR
17-O-Acetylnorajmaline
Ajmalicine
Secologanin
Strictosidine synthase (STR)
VR2
19,20-
a-(S)-dihydrovomilenine
AAE
Strictosidine aglycoside
SDG
4,21-dehydrogeissoschizine
Cathenamine
Yohimbine
FIGURE 8.2
Enzyme-catalyzed biosynthesis of reserpine alkaloids via strictosidine intermediate.
Peroxidase
Serpentine
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8.4.3 Biosynthesis of strictosidine
The biological material can be the key to successfully investigate a biosynthetic
pathway at the molecular level. This is especially true for pathways operating in
higher plants due to their slow growth characteristics. This “upstream pathway”
was elucidated in detail on enzymatic and genetic level (Geu-Flores et al., 2012;
Asada et al., 2013; Salim et al., 2013, 2014; Miettinen et al., 2014). However, the
manifold reactions, spearheaded by deglucosylation of strictosidine, providing the
abundance of MIA carbon skeletons, are still elusive. Further, of the various “downstream pathways” leading to the pharmacologically important structures, only a few
are known. This condensation is catalyzed by the enzyme STR (Treimer and Zenk,
1979) through a stereoselective PicteteSpengler reaction mechanism in order to
yield a b-carboline product (Mar esh et al., 2008). Strictosidine b-glucosidase (Lui-
jendijk et al., 1998) cleaves the glucose moiety of strictosidine to produce an unsta-
ble aglycone molecule which spontaneously leads to the formation of a series of
reactive intermediates that serve as starting materials for the biosynthesis of different
MIA backbones/groups.
8.4.4 Ajmaline
Ajmaline is a class I antiarrhythmic agent, it is highly useful in diagnosing Brugada
syndrome (hereditary card iac disorder) (Brugada et al., 2003), and differentiating
between subtypes of patients with this disease (Paul et al., 2003). These agents
are primarily classified into four major groups on the basis of their mechanism of
action, i.e., sodium channel blockade, beta-adrenergic blockade, repolarization prolongation, and calcium channel blockade. Ajmaline is a sodium channel blocker that
shows instant action when given intravenously, which makes it ideal for diagnostic
purposes (Dobbels et al., 2016). It has been reported to stimulate respiration and intestinal movements. The action of ajmaline on systemic and pulmonary blood pressure is similar as of serpentine.
8.4.5 Biosynthesis of ajmaline
Simplified pathway leading from strictosidine via vomilenine to ajmaline. Two
routes from vomilenine to 17-O-acetylnorajmaline can be postulated, depending
on which of the two double bonds is reduced, first: reduction of the indolenine
ring in 1,2-position (VR) followed by reduction of the 19,20-double bond
(DHVR), as proposed by Gao et al. (2002) and von Schumann et al. (2002). The
next step along the ajmaline biosynthetic pathway involves hydrolysis of the 17O-acetylated norajmaline by the enzyme acetylajmalan esterase (AAE). The last
step involves an S-adenosyl-
et al., 1983) which catalyzes the indoline nitrogen methylation of norajmaline to
produce ajmaline.
L-methionineedependent methyltransferase (Sto
ckigt
¨
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8.4.6 Biosynthesis of serpentine
Serpentine are indole alkaloids of considerable medicinal importance. Serpentine is
an inhibitor of topoisomerase (Type II) as well as antipsychotic properties (Dasson-
neville et al., 1999; Santos et al., 2017). In vacuole, an enzyme (PER) peroxidase is
responsible for oxidation of ajmali cine to serpentine (O’Connor and Maresh, 2006).
8.4.7 Biosynthesis of yohimbine
Yohimbine is an indoloquinolizidine alkaloid, used as a selective alpha-adrenergic
antagonist or alpha-blocker in the blood vessels for the treat ment of erectile dysfunction in man (Goldberg and Robertson, 1983; Morales, 2000). Its action on peripheral
blood vessels is weaker as compared to reserpine. Yohimbine has a mild antidiuretic
action, probably via stimulation of hypothalmic center and release of posterior pituitary hormone. Antagonism at these receptors relaxes smooth muscle and lowers
blood pressure.
Biosynthetic pathway of Yohimbine is involved homoallylic isomerization of the
keto dehydrogeissoschizine followed by 1,4 conjugate addition. However, detailed
mechanism of biosynthetic route of Yohimbine is yet to be identified due to unidentification of enzymes in deglycosylated strictosidine.
8.5 Omics strategies and advancements
Plants are a rich source of assorted specialized metabolites that have been utilized
for thousands of years as scents, flavoring agents, pigments, insect repellents, and
therapeutic compounds (Facchini et al., 2012). These secondary metabolites are
considered to be the fundamental approach used by plants to acclimate and persist
in various ecological niches and environmental conditions and to tackle biotic as
well as abiotic intrusions in their natural habitat (Furstenberg-Hagg et al., 2013;
Weng, 2014).
The specialized plant secondary metabolites are characterized by complex chemical structures which are derived from simpler precursors, suggesting the involvement of complicated biosynthetic machinery and regulatory processes that have
been strongly favored and developed via natural selection (Moore et al., 2014).
Therefore, mapping plant genes to a biosynthetic pathway necessitates the
tedious and time-consuming experimental approach of figuring out one gene at a
time. Such identification becomes even more challenging due to genetic redundancy
and tight genetic regulation; therefore, to confirm or verify the function of a plant
gene, multiple lines of evidence are required. Since secondary metabolic pathways
and their regulation include extremely complex frameworks with interconnected
components, studies comprising association-based analysis within multiple elements can serve as an important alternate for plant-derived metabolic pathway discovery (Steuer, 2007; Sweetlove et al., 2008; Tomar and De, 2013).
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In recent years, “Omics”-based strategies have gained a great deal of popularity
among plant biologists and research community as an approach for functional characterization of target plant genes and to investigate systems response under specific
conditions (Muranaka and Saito, 2013; Saito, 2013; Wurtzel and Kutchan, 2016).
Qualitative and quantitative analysis of various elements of a biological system,
such as specialized metabolites, transcript expression, and protein levels while
capturing the spatiotemporal responses, provides imperative insights into various
ongoing processes and interactions/associations within them. While each omics
datasets gives a broad overview of the static or dynamic condition of a biological
system, the integration of various datasets provides an effective and efficient means
to strengthen genuine observations and reduces the probability of false positives/
negatives (Moreno-Risueno et al., 2010; Deshmukh et al., 2014; Rai et al., 2016).
The representation of different omics approaches and strategies used in
R. serpentina has been depicted in Fig. 8.3.
8.5.1 Genomic technologies and genetic markers
Whole-genome sequences serves as an imperative resource for understanding the total biosynthetic potential of a medicinal plant. It also facilitates the development of
herbal medicines and selection of cultivars with desired agricultural traits and high
levels of secondary metabolites having pharmaceutical and medicinal importance
(Hao and Xiao, 2015; Unamba et al., 2015). Total reliance on medicinal plan ts for
extraction of essential bioactive compounds has proved to be an impractical and unsustainable approach (Chang and Keasling, 2006; Facchini et al., 2012); therefore,
FIGURE 8.3
The representation of different omics approaches and strategies used in Rauwolfia
serpentina.
8.5 Omics strategies and advancements 123
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knowledge and understanding of biosynthetic pathway components offers an opportunity to develop alternate sources for obtaining these important compounds.
Genomic sequences give vital information on plant origin and evolution, inheritable characters, physiological and developmental schematics, epigenetic regulation,
and metabolic potential, which serves as the basis for interpreting genetic as well as
chemodiversity at the molecular level (Dhanapal and Govindaraj, 2015; Hao and
Xiao, 2015; Unamba et al., 2015).
Genomic sequences helps in the development of easily available and robust functional genomic resources, like full-length complementary DNA (cDNA) clones,
tagged mutant lines, and facile and rapid transformation method. It also provides
essential information on multiple homologous genes of specialized biosynthetic
pathways, thereby improving capability and possibilities to carry out gene knockout
experiments to decipher their functionality.
Elucidation of the MIA biosynthesis has recently advanced in Apocynaceae family through simultaneous development of transcriptomic resource analyses and
reverse genetics strategies achieved by means of virus-induced gene silencing
(VIGS). Most of these tools have been basically adapted for Catharanthus roseus;
however, the VIGS technique has been barely used on other Apocynaceae species.
Rauwolfia species not only constitutes a significant source of explicit and valuable
secondary metabolites such as reserpine, ajmaline, ajmalicine, etc., but are also
well-established models for understanding alkaloid metabolism, and as such would
certainly benefit from an effective VIGS procedure. It has been demonstrated that
biolistic-mediated VIGS technique can be efficiently used for gene silencing in
both R. serpentina and R. tetraphylla taking advantage of a recently modified inoculation method in tobacco rattle virus (TRV) vectors via particle bombardment (Cor-
bin et al., 2017). TRV vectors, namely pTRV1 and pTRV2-MCS encoding the two
genomic components of TRV obtained from Arabidopsis Biological Resource
Centre (http://www.arabidopsis.org), were used to generate silencing constructs
and for propagating the virus within Rauwolfia plantlets. After standardizing
bombardment conditions while minimizing transformed plantlet injury, gene downregulation was observed with an approximately 70% decr ease in expression by
silencing phytoene desaturase gene in both Rauwolfia species. This established
gene silencing methodology will thus contribute as a valuable tool in identification
and characterization of alkaloid biosynthesis genes in these prevalent Rauwolfia species as well as other closely related medicinal plant species.
DNA-based molecular markers have been widely utilized in recent years for
assessment of genetic diversity among the germplasm in various medicinal plant
species. Nair et al. (2014) investigated genetic diversity in populations of
R. serpentina based on random amplified polymorphic DNA (RAPD) marker s.
Within population a high genetic diversity and among population high genetic differentiation was revealed which was suggested to be caused both by habitat fragmentation of the lower size populations as well as the low gene flow level among
them. The findings of this study showed that RAPD-based assessment of genetic diversity in R. serpentina seemed to be adequately informative and powerful. The
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information on genetic diversity and structure among populations of R. serpentina
would be helpful in developing appropriate conservation strategies and breeding
programs.
Comparative genomics has been described as one of the most effective and successful methods for characterization of gene functions, identification of biomarkers,
and investigation of evolutionary relatedness in humans (Moreno et al., 2008)as
well as plants (Moreno et al., 2008; Michael and Jackson, 2013). Pathania et al.
(2016) carried out comparative co-expression analysis of R. serpentina and
C. roseus which revealed evolutionary complexity in secondary metabolism.
Comparative analysis approach is instigated for comparing two or more organisms
in order to identify similarities among them as well as to investigate mechanisms
responsible for diversification in various key biological phenomena such as photosynthesis, reproduction and defense response, metabolic pathways, and many
more. The basis of comparative analysis is that biologically significant processes
remain conserved across different organisms in comparison to nonrelevant associations which decline with evolutionary time scale (Hansen et al., 2014). Comparative
co-expression analysis technique can be used to determine genes playing role in differential tissue-specific and species-specific biosynthesis of metabolites. Highthroughput expression data availability and the use of computational analysis
approach for integration of expression data prompted to determine candidate
genes/biomarkers involved in variation of MIAs between R. serpentina and
C. roseus. Network-based approach was used to carry out differential expression
analysis for identification of candidate genes accountable for species-specific production of metabolites in these medicinal plants. The key genes of MIA biosynthesis
contributing toward diversification of metabolites have been identified using this
approach.
Gene expression is considered as an intricate phenomenon being regulated by a
set of proteins known as transcription factors (TFs), which are responsible for activating or repressing various genes (Mitsuda and Ohme-Takagi, 2009). This gene
regulation by TFs takes place via a set of highly synchronized internal and/or
external signals. Some TFs also interact with one another to regulate genes (Yang
et al., 2012). Identification of TFs regulating secondary metabolism in
R. serpentina has been carried out by Pathania and Acharya (2016). Unraveling
the interaction between genes and various TFs is essential for gaining complete understanding and knowledge of secondary metabolism in plants. It is necessary to
identify transcriptional regulators along with their targets (genes) involved in secondary metabolites bios ynthesis, to gain in-depth insights of metabolic pathways
associated with them. The authors employed an integrative appro ach using omics
data in order to identify TFs with unknown functionality and illustrated their roles
in regulating valuable metabolites along with metabolic traits. Identification of
TFs functionality was carried out by implementin g gene co-expression network
analysis, which could not have been possible to annotate using any other simple
methods. The TF families, WRKY and AP2-EREBP, were identified to be playing
regulatory roles in regulating alkaloids biosynthesis in R. serpentina. TFs regulating
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